International Journal of Nanomedicine
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Open Access Full Text Article
Antibacterial activity of trimetal (CuZnFe) oxide
nanoparticles
Khalid E Alzahrani 1,2
Abdurahman A Niazy 3
Abdullah M Alswieleh 4
Rizwan Wahab 5
Ahmed M El-Toni 2
Hamdan S Alghamdi 3
1
Department of Physics and
Astronomy, King Saud University,
Riyadh, Kingdom of Saudi Arabia;
2
King Abdullah Institute for
Nanotechnology, King Saud University,
Riyadh, Kingdom of Saudi Arabia;
3
Prince Naif Health Research
Center, Molecular and Cell Biology
Laboratory, College of Dentistry, King
Saud University, Riyadh, Kingdom
of Saudi Arabia; 4Department of
Chemistry, College of Science, King
Saud University, Riyadh, Kingdom
of Saudi Arabia; 5Department of
Zoology, College of Science, King
Saud University, Riyadh, Kingdom
of Saudi Arabia
Correspondence: Khalid E Alzahrani
Department of Physics and Astronomy,
King Abdullah Institute for
Nanotechnology, King Saud University,
PO Box 2455, Riyadh 11451, Kingdom
of Saudi Arabia
Tel +966 55 300 1262
Email alzkhalid@ksu.edu.sa
Hamdan S Alghamdi
Molecular and Cell Biology Laboratory,
College of Dentistry, King Saud
University, PO Box 2455, Riyadh 11545,
Kingdom of Saudi Arabia
Tel +966 50 341 8405
Email dalghamdi@ksu.edu.sa
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http://dx.doi.org/10.2147/IJN.S154218
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O r i g in a l R e s e a r c h
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Background: The increasing resistance of pathogenic bacteria to antibiotics is a challenging
worldwide health problem that has led to the search for new and more efficient antibacterial
agents. Nanotechnology has proven to be an effective tool for the fight against bacteria.
Methods: In this paper, we present the synthesis and traits of trimetal (CuZnFe) oxide nano-
particles (NPs) using X-ray diffraction, high-resolution transmission electron microscopy, and
energy dispersive x-ray spectroscopy. We evaluated the antibacterial activity of these NPs against
gram-negative Escherichia coli and gram-positive Enterococcus faecalis and then compared it
to that of their pure single-metal oxide components CuO and ZnO.
Results: Our study showed that the antibacterial activity of the trimetal oxide NPs was greater
against E. coli than against E. faecalis. Overall, the antimicrobial effect of trimetal NPs is
between those of pure ZnO and CuO nanoparticles, which may mean that their cytotoxicity is
also between that of pure ZnO and CuO NPs, making them potential antibiotics. However, the
cytotoxicity of trimetal NPs to mammalian cells needs to be verified.
Conclusion: The combination of three metal oxide NPs (ZnO, CuO, and Fe2O3) in one multim-
etal (CuZnFe) oxide NPs will enhance the therapeutic strategy against a wide range of microbial
infections. Bacteria are unlikely to develop resistance against this new NP because bacteria
must go through a series of mutations to become resistant to the trimetal oxide NP. Therefore,
this NP can combat existing and emerging bacterial infections.
Keywords: nanotechnology, antibacterial agents, nanoparticles, trimetal nanoparticles,
Escherichia coli, Enterococcus faecalis
Introduction
Bacterial infections are major challenges for the medical field and have become a
serious threat to human health and lives. An increasing number of species of bac-
teria have developed resistance against most common antibiotics from the misuse
of these drugs.1 The list of drug-resistant bacteria has increased dramatically, for
example, Enterobacter cloacae is sulfonamide- and methicillin-resistant, Staphy-
lococcus aureus is vancomycin-resistant, Mycobacterium tuberculosis is resistant
to multiple drugs, and Streptococcus pyogenes and Acinetobacter baumannii are
macrolide-resistant.2–4 Generally, biofilm-growing bacteria are highly resistant to
antibacterial drugs and the host immune system, although the exact mechanisms
underlying such resistance are still not fully understood.5,6 Biofilm is a structured
community of bacteria embedded in a self-produced extracellular matrix of proteins,
polysaccharides, and DNA. Consequently, infections involving biofilm formation
are chronic and difficult to treat. Therefore, there is an urgent need to find alterna-
tive therapeutic approaches for overcoming the increasing resistance of bacteria to
current antibiotics.
International Journal of Nanomedicine 2018:13 77–87 77
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Alzahrani et al
Controlling the growth of bacterial biofilms using nanopar-
ticles (NPs) has become the center of attention in the medical
field. The use of NPs is a promising therapeutic strategy for
overcoming the increasing emergence of multidrug-resistant
bacteria. Although the antimicrobial effects of NPs have been
verified using different types of NPs on various bacterial spe-
cies, the bactericidal mechanisms of NPs are still being inves-
tigated. Several factors such as the physiochemical properties
of NPs and the bacterial species involved might play important
roles in the antibacterial activity of NPs. Some bacterial species
are more sensitive to particular NPs than are others. Ag NPs
are more efficient than Cu NPs against Escherichia coli and
S. aureus, whereas Bacillus subtilis showed more susceptibility
to Cu NPs than to Ag NPs.7 Titanium dioxide NPs exhibited
greater antibacterial activity against E. coli than against
Salmonella typhimurium.8 Dizaj et al9 showed that Ag NPs
have a greater antibacterial effect on Streptococcus mutans
than do ZnO and Au NPs. The sensitivity of bacteria to NPs is
related to the species whereby the cell structure of the bacteria
influences its tolerance to NPs. Vancomycin-resistant bacte-
ria (eg, Enterococci) develop an additional outer membrane
that covers the cellular surface and protects the bacterium
from the vancomycin. Vancomycin-capped Au NPs can pen-
etrate the outer cell membrane, allowing the vancomycin to
access the cellular surface.10 Poly(vinyl alcohol)-coated ZnO
NPs can kill bacterial cells by inducing oxidative stress.11
Furthermore, the size of the NP plays an important role in its
toxicity. Hayden et al12 studied the effect of size reduction on
the antibacterial efficiency of Au NPs and found that 2 nm Au
NPs are more toxic to B. subtilis than 6 nm Au NPs.
Several types of metal and metal oxide NPs such as CuO,
CaO, Ag and Ag2O, Au, ZnO, and MgO have been investigated
for their antibacterial effects. Diverse studies revealed that
ZnO NPs exhibit a wide spectrum of antibacterial activities
toward various gram-negative and gram-positive bacteria.13
For example, they are very efficient at inhibiting the growth of
gram-negative E. coli and Pseudomonas aeruginosa and gram-
positive S. aureus and B. subtilis.14–17 The antibacterial effect
of ZnO NPs on S. mutans was compared with that of other
NPs such as Ag and Au and it was observed that ZnO NPs had
the lowest bactericidal activity.18 However, many toothpastes
contain Zn to combat dental plaque that is formed by bacteria
such as S. mutans.19 The antimicrobial activities of ZnO NPs
have been found to be affected by the size and concentration
of the NPs but the bactericidal mechanisms of Zn and ZnO
NPs still are unknown. Recently, ZnO NPs have begun to
be used in food packaging, although there is some concern
about the potential impact of NPs on the health of consumers.
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According to some studies, ZnO NPs exhibit minimal toxicity
to human cells.20 Heng et al21 demonstrated the cytotoxic effect
of ZnO NPs on human bronchial epithelium cells, but the effect
of ZnO NPs on other human cells has not been confirmed yet
and additional research is needed. In addition, Ingle et al22
has reported interesting data on the antimicrobial effect of Cu
and CuO NPs. Das et al23 demonstrated that nanostructured
Cu efficiently inhibits the growth of S. mutans, E. coli, and B.
subtilis. Xu et al24 studied the susceptibility of gram-positive
and gram-negative bacteria to Cu NPs and found that gram-
negative bacteria are more susceptible. Like other NPs, Cu NPs
showed size-dependent bactericidal activity. Several in vivo
and in vitro studies on the toxicity of Cu NPs to human cells
showed that they can cause irreversible damage.25–27
The main aim of our study was to combine the strong
antibacterial activity of ZnO and CuO NPs with that of Fe2O3.
ZnO and CuO NPs showed the most antibacterial activity
against various bacterial species, while Fe2O3 NPs showed
the least toxicity.17 The combination of the three metal oxide
NPs formed a new nanostructured CuZnFe oxide NP, called a
trimetal oxide NP. To our best knowledge, multi-metal oxide
NPs are usually prepared using complicated methods.28 In the
present study, we report, for the first time, the synthesis of
CuZnFe oxide NPs using inexpensive chemicals and very
easy solution process to form good quality nanostructures for
large-scale production. This composite NP has the potential
to control a wider range of bacterial infections compared to
single NP material. Although ZnO NPs are relatively less toxic
to human cells than CuO NPs in aqueous solution, they tend to
aggregate and form large flocculates that decrease their anti-
bacterial activity.29 The stability of ZnO NPs in aqueous media
may be improved by combining them with Fe2O3 NPs. Fe2O3
may provide some magnetic properties to these NPs resulting
in their use in several additional applications. We tested the
antibacterial activities of the CuZnFe oxide NP against gram-
negative E. coli and gram-positive Enterococcus faecalis.
Bacteria are unlikely to develop resistance against this new
NP because bacteria must go through a series of mutations to
become resistant to the trimetal oxide NP. Therefore, this NP
can combat existing and emerging bacterial infections.
Materials and methods
Synthesis of ZnO NPs
ZnO NPs were synthesized using zinc acetate dihydrate
[Zn(CH3COO)2•2H2O] and n-propyl amine [CH3-(CH2)2-
NH2], purchased from Sigma-Aldrich Corporation (St Louis,
MO, USA) and used without further purification. In a typical
experiment, ~20 mL of n-propyl amine and 0.3 M zinc acetate
International Journal of Nanomedicine 2018:13
Dovepress
dihydrate were dissolved in 100 mL of methanol (MeOH).
The obtained solution was stirred for 30 min for complete
dissolution. The pH of the solution, measured using an
expandable ion analyzer (Cole Parmer, Vernon Hills, IL,
USA), was 12.2. After complete dissolution, the mixture was
transferred to a three-necked refluxing pot and refluxed at
65°C for 6 h. After 6 h, a white precipitate was observed in
the double-necked refluxing pot. A specialized temperature
controller measured and controlled the refluxing temperature.
The white powder was washed several times with MeOH,
ethanol (EtOH), and acetone and dried at room temperature
in a glass Petri dish. The obtained powder was characterized
by its structural and chemical properties.
Synthesis of CuO NPs
CuO NPs were synthesized using copper acetate hydrate
[Cu(CH3•COO)2•H2O], n-propyl amine [CH3-(CH2)2-NH2],
and sodium hydroxide (NaOH), purchased from Sigma-
Aldrich and used as received. In a typical experiment, 0.3 M
of copper acetate hydrate and 20 mL of n-propyl amine were
mixed in 100 mL of MeOH with constant stirring, after which
the solution turned blue. NaOH (0.1 M) was mixed into the
solution in steps and shaken each time to ensure complete mix-
ing. After adding the NaOH, the pH of the solution, checked
via a pH meter (Cole Parmer), was 12.01 because of the
increased basicity of the solution. The solution was transferred
to a double-necked refluxing pot and refluxed at ~90°C for 6 h.
As the temperature of the solution increased, the color changed
from blue to dark brown to black. After the reaction was
complete, the product was centrifuged at 3,000 rpm for 3 min
and washed repeatedly with MeOH, EtOH, and acetone to
remove the intermediate by-products. The material was dried
at room temperature in a glass Petri dish and utilized for fur-
ther chemical, morphological, and biological studies.
Formation of CuZnFe oxide NPs
Trimetal CuZnFe oxide NPs were formed by dissolving
0.3 M of zinc acetate dihydrate [Zn(CH3COO)2⋅2H2O], 0.3 M
of copper acetate hydrate [Cu(CH3COO)2⋅H2O], and 0.3 M
of iron nitrate nonahydrate [Fe(NO3)3⋅9H2O] in 300 mL of
MeOH and stirring constantly for 30–40 min. When the solu-
tion was completely dissolved, 60 mL of n-propyl amine
[CH3-(CH2)2-NH2] was added, followed by the dropwise
addition of 0.1 M of NaOH to increase the basicity of the
solution. The pH of the solution, checked via a pH meter
(Cole Parmer), was 12.61. The mixture was transferred to
a double-necked refluxing pot and refluxed at ~90°C for
6 h to obtain a red precipitate. The precipitate was washed
International Journal of Nanomedicine 2018:13
Trimetal (CuZnFe) oxide nanoparticles
several times with MeOH, EtOH, and acetone to remove ionic
impurities and dried at room temperature. The material was
studied for its structural and chemical properties.
Characterization of NPs
The phase, crystallinity, and size of the prepared nanostruc-
tures were characterized by their x-ray diffraction (XRD)
pattern (Rigaku, Tokyo, Japan) obtained with CuKα radia-
tion (λ=1.54178 Å) in the 20°–80° range at a scan speed
of 6°/min, an accelerating voltage of 40 kV, and current
of 40 mA. The morphology of the prepared nanostructures
was analyzed using transmission electron microscopy
(TEM, 200 kV; Hitachi, Tokyo, Japan). For TEM analysis
of the nanostructures, NP powder was sonicated in EtOH
for 10–20 min. Then a C-coated Cu grid (400 mesh) was
dipped into the solution and dried at room temperature for
morphological analysis.
In vitro experimental procedures
For the gram-positive bacterium, we chose E. faecalis
because it can cause life-threatening infections in humans
and shows a high level of resistance to antibiotics. In addi-
tion, E. faecalis has been found to be associated with up to
90% of the cases of chronic infection in teeth that underwent
root canal treatment. For the gram-negative and facultative
anaerobic bacterium, we chose E. coli, which can cause
serious food poisoning and is occasionally responsible for
food contamination.
Cell death analysis
The method used to measure bacterial cell death was previ-
ously described.30,31 Briefly, cell death of E. faecalis and
E. coli was analyzed using fresh culture grown in 25 mL
of nutrient broth (NB; yeast extract 2 g/l, peptone 5 g/l,
NaCl 5  g/l) in a 50-mL conical flask until the stationary
phase showed no cell growth. The density of E. faecalis and
E.  coli was measured using an Eppendorf BioPhotometer
Plus spectrophotometer (Thomas Scientific, Swedesboro,
NJ, USA). A portion (5 mL) of the culture was transferred
to a new sterile 20 mL conical flask. The bacteria were then
added to three conical flasks containing Zn, Cu, and CuZnFe
oxide NPs at 150 µg/mL and to three control flasks that did
not contain any NPs. The flasks were then placed in a shak-
ing incubator and shaken at 220 rpm for 5 h at 37°C, after
which 1 mL from each flask was used to measure the optical
density at 595 nm. The blank flasks contained 150 µg/mL
of the corresponding NPs and media. The average of three
readings was used for each NP.
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Alzahrani et al
Biofilm formation
The aim of this part was to test the ability of the bacteria
E. faecalis and E. coli to form a biofilm in the presence of
CuO, ZnO, and CuZnFe oxide NPs. Microbial biofilm is
the main method of overcoming a harsh environment and
the host immune system.30 We used the spectrophotometric
assay method to study biofilm formation. The bacteria were
grown in a 96-well plate in triplicate using NB. Each well
contained either NB with no NPs or NB with ZnO, CuO, or
CuZnFe oxide NPs at 150 µg/mL. The plate was then incu-
bated at 37°C for 48 h after being tightly sealed to prevent
dehydration. The media in the plate were then poured into
a designated waste container with 10% bleach. The plate
was submerged in water to eliminate any excess media and
was left to air dry. One-hundred twenty-five microliters of
0.1% crystal violet solution was added to each well and the
plate was incubated at room temperature for 10 min. The
crystal violet solution was poured into the designated waste
container. The plate was submerged in water to eliminate any
excess solution and left to air dry. Next, 200 µL of 100%
EtOH was added to each well and the plate was incubated at
room temperature for 15 min. EtOH in each well was mixed
by pipetting gently to evenly distribute the color. The plate
was then read in a Synergy™ HT Multi-Mode microplate
reader (BioTek, Winooski, VT, USA) at λ=490 nm.
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CFU/mL analysis
To test the ability of E. faecalis and E. coli to grow in the pres-
ence of the NPs, a viable cell count per milliliter was conducted
using the colony-forming unit (CFU)/mL method. Bacterial
cells (1 mL), grown overnight in 20 mL of NB (grown in a
sterile 50 mL conical flask) and standardized to 1×105 cells,
were mixed with 150 µg of ZnO, CuO, and CuZnFe oxide
NPs and then the mixture was added to 9 mL of sterile distilled
water. The solution underwent serial dilution until a dilution of
1×10-7 was obtained. NP-exposed samples and a control were
streaked on four NB Petri dish plates each. The plates were
then incubated overnight at 37°C. Each sample was tested in
triplicate and the average of the three results was used.
Statistical analysis
To compare the quantitative variables of the groups, we
used the nonparametric Mann–Whitney test; p,0.05 was
considered statistically significant.
Results
Characterization of CuZnFe oxide NPs
We used the XRD pattern, shown in Figure 1, to characterize
the size, phase, and crystalline character of CuZnFe, ZnO,
and CuO NPs. Comparison of the XRD patterns, in Figure 1,
to the XRD patterns of the materials in the International















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Figure 1 X-ray diffraction spectra of ZnO, CuO, and CuZnFe oxide nanoparticles. *Indicates that the peak is unidentified.

80 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2018:13

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Figure 2 Scanning electron micrograph of the CuZnFe oxide nanoparticles.
Center for Diffraction Data identified our ZnO (black line),
CuO (red line), and the trimetal CuZnFe oxide NPs (blue
line) as ZnO (JCPDS card No 36–1451), CuO (JCPDS
card No 05–066), and trimetal CuZnFe oxide (JCPDS card
No 07–0392), respectively. However, three unidentified
peaks were observed on the XRD spectrum of the CuZnFe
oxide NPs. These peaks can be attributed to the complex
formation of nanostructures in the mixed composite. The
size of the crystallite can be calculated from XRD spectra
using the Scherrer formula; D = kλ/β cosθ. The average size
of the crystallite of the CuZnFe oxide NPs was calculated to
be 23 nm, where k=0.9, λ=0.1541, and β is the full width at
half-maximum of the peak at θ.
The morphology of the CuZnFe oxide NPs was character-
ized using scanning electron microscopy (SEM) and high-
resolution TEM (HRTEM). Representative images from
SEM and HRTEM are shown in Figures 2 and 3, respectively.
The CuZnFe oxide NPs are almost spherical and have a
clear tendency to agglomerate, which is normal for NPs
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Figure 3 (A) High-resolution transmission electron microscope micrograph of the mixed CuZnFe oxide nanoparticles (NPs). (B) Atomic resolution of mixed CuZnFe oxide NPs.
International Journal of Nanomedicine 2018:13
Trimetal (CuZnFe) oxide nanoparticles
because of the strong interparticle interaction induced by
high surface energy. The size of agglomerate was analyzed
by dynamic light scattering (Zeta Nanosizer) and found to
be in 128 nm range. The average size of CuZnFe oxide NPs,
based on several HRTEM images taken at different angles,
was estimated to be 42±2 nm, whereas the average particle
size based on XRD measurements was 19 nm smaller. The
smaller size may be the result of the nanostructures merging
together to form a larger molecule of mixed nanostructures.
This is indicative of the presence of particle agglomeration,
which is clarified by the HRTEM images of lattice fringes
(Figure 3). The XRD calculation represents only the average
single particle dimension for the bulk/powder nanostructures.

:'PP The nanostructures were further assessed using energy
dispersive x-ray spectroscopy (EDX), which is usually
employed to map NPs based on their composition. Figure 4
shows the EDX spectrum of the mixed CuZnFe oxide NPs.
The spectral peaks correspond to Zn Cu, Fe, and O, con-
firming the presence of elemental Zn, Cu, and ferrite, with
O, in the NPs. No other impurity related to other elements
was exhibited in the spectrum, which further shows that the
synthesized nanostructures were only the mixed metal oxide
nanostructures used.
Antibacterial activity of CuZnFe
oxide NPs
Metallic NPs like Ag, Cu, Au, Ti, and Zn have proven effec-
tive against a wide range of bacterial species. Zn and Cu are
widely used to combat microbial infections and have shown
relatively less cytotoxicity.32 The size of NPs allows them to
penetrate the cell wall of bacteria and interact with cellular
elements.33 Therefore, the size of NPs may contribute to their
bactericidal effect. The combination of the three metal oxides
in the CuZnFe oxide NPs may enhance the therapeutic abili-
ties of the NPs against a wide range of microbial infections.
QP
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Alzahrani et al Dovepress





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Figure 4 Energy dispersive x-ray spectroscopy spectrum of mixed CuZnFe oxide nanoparticles.
Figure 5 compares the bactericidal effect of the trimetal
(CuZnFe) oxide NPs used in this study with that of pure ZnO
and CuO NPs. The effect of FeO NPs against the bacterial
cells was not investigated in this study. FeO NP has been
proven to have a low or no toxic effect on different bacterial
species, in particular E. coli.17 The minimum concentration
needed for the metal oxides to have a bactericidal effect
on both bacterial species was at 150 µg/mL. The cells not
exposed to NPs showed no drop in cell density after 5 h of
incubation, while cells exposed to NPs showed a significant
drop in cell density, except E. coli (gram-negative) exposed
to CuO, where there was no cell death recorded. However,
E. coli showed a 55% drop in cell density with exposure to
ZnO and a nearly 85% drop in cell density with exposure
to CuZnFe oxide NPs. E. faecalis (gram-positive) had the
greatest drop in cell density when exposed to CuO NPs, with
an ~59% drop in live cells compared to an ~25% drop in live
cells with ZnO exposure and a 55% drop with exposure to
trimetal (CuZnFe) oxide NPs.
The effect of trimetal (CuZnFe) oxide NPs on biofilm
formation by both bacteria was proved, as shown in Figure 6.
However, CuZnFe oxide NPs had the least effect on biofilm
formation when compared to that of ZnO and CuO. There
was an ~50% reduction in biofilm formation by E. coli in the
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presence of CuZnFe oxide NPs, while the presence of ZnO
and CuO NPs caused an ~65% drop in biofilm formation
by E. coli compared to control group. E. faecalis showed
only a 26% drop in biofilm formation in the plate contain-
ing CuZnFe oxide NPs but a 62% and 40% drop in biofilm
formation with ZnO and CuO NPs, respectively.
The growth of E. faecalis and E. coli on a nutrient agar
was observed directly using a light microscope (Figure 7).
This method is a useful and simple way to observe overnight
growth of microbes without the presence of NPs or with
150 µg/mL of ZnO, CuO, or CuZnFe oxide NPs, and their
effect on the CFU/mL count. Figure 8 presents the mea-
surements of CFUs, indicating the antibacterial activity of
CuZnFe oxide NPs. The viability of E. faecalis and E. coli
was reduced by 40% and 38%, respectively, in the pres-
ence of CuZnFe oxide NPs. However, CuO NPs had almost
no effect on E. coli, whereas they reduced the viability of
E. faecalis by 70%. E. faecalis and E. coli showed a decrease
in cell viability of 22% and 75%, respectively, after being
exposed to ZnO NPs.
Discussion
Trimetallic nanoparticles were successfully synthesized
by combining ZnO and CuO NPs with that of Fe 2O 3.
International Journal of Nanomedicine 2018:13
Dovepress Trimetal (CuZnFe) oxide nanoparticles

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Figure 5 Percentage of live (A) Enterococcus faecalis (E. faecalis) cells and (B) Escherichia coli (E. coli) cells after 5 h of exposure to 150 µg/mL of metal oxide nanoparticles.
Cells were grown in nutrient broth.

The combination of the three metal oxide NPs formed a new
nanostructured CuZnFe oxide NP. In the present study, we
used inexpensive chemicals and a very easy process for fab-
rication of high-quality trimetal (CuZnFe) oxide NPs. Multi-
metal oxide NPs are usually prepared using complicated
methods. Based on the synthesis of CuZnFe oxide NPs, the
use of metal salt of acetate and amine, in the current study,
was due to that the acetate and amine can be easily dissoci-
ated in solution due to the breaking of hydrogen bonds at the
time of refluxing of solution resulting in reaction with the
other metal compound. This will easily react with the metal
acetate (MCH3COO−) group initially. In solution, the metal
ions (M=Cu2+, Fe2/3+ and Zn2+ ions) react with n-propyl-amine
group to form a metal complex (MN-(CH2)2-CH3). When
the refluxing temperatures increase, the hydroxyl (OH−) ions
from the used solvent methanol react with this metal complex
and the metal ions from the complex decomposes, and then
react to the hydroxyl (OH−) ions to form metal hydroxide
[M(OH)2]. At higher refluxing temperature, hydroxide
molecules change into metal oxide and water molecules.
The by-products of the reaction [(CH3)2N-(CH2)2-CH3,
CH3COOH and water molecule] were leached out during
centrifugation of the product.34,35
Antibacterial activities of CuZnFe oxide NPs were tested
against gram-negative E. coli and gram-positive E. faecalis.
CuZnFe oxide NPs had an effect on both bacterial cells by
reducing their viability and their ability to synthesize biofilm.
Over the last few decades, bacterial resistance has challenged
scientists to develop novel antimicrobial agents. Among
them, metal NPs have shown strong antibacterial activity in
several studies.2,3,9 Importantly, it is hypothesized that metal
NPs might have the potential to control bacterial resistance
because NPs target multiple biomolecules at once.8,9 How-
ever, the effect of metal oxide NPs on bacterial growth varies
widely among bacterial species and even among strains
of the same species.7,17,20 Based on the cell wall structure,
bacterial cells can be divided into two distinct types: gram-
positive and gram-negative. Gram-positive bacteria have a
thicker layer of peptidoglycan in their cell wall than that of
gram-negative bacteria. However, gram-negative bacteria

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83
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Alzahrani et al Dovepress

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Figure 6 Ability of (A) Enterococcus faecalis (E. faecalis) and (B) Escherichia coli (E. coli) in nutrient broth to form biofilm after incubation with 150 µg/mL of metal nanoparticles
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Figure 7 Representative images of overnight growth of microbes on nutrient agar with no nanoparticles (NPs) or with 150 µg/mL of ZnO, CuO, or CuZnFe oxide NPs and
their effect on the colony-forming unit/mL count: (A) Enterococcus faecalis, (B) Escherichia coli.

84 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2018:13

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Dovepress Trimetal (CuZnFe) oxide nanoparticles

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&RQWURO &X =Q &X=Q)H

Figure 8 Colony-forming units (CFU) for (A) Enterococcus faecalis (E. faecalis) and (B) Escherichia coli (E. coli) after incubation in nutrient broth overnight with no nanoparticles
(NPs) or in the presence of 150 µg/mL of metal oxides NPs.

possess an additional outer membrane constructed mainly of
negatively charged lipopolysaccharide molecules (LP).
In the present study, we observed that CuZnFe oxide
NPs have detrimental effects on E. coli (gram-negative)
compared to the effects caused by individual CuO and ZnO
NPs. CuZnFe oxide NPs were also found to be more bacte-
ricidal than ZnO against E. faecalis (gram-positive), but 7%
lower than CuO NPs. CuZnFe oxide NPs in this study form
agglomerates with an average size of ~128 nm. It seems that
these large agglomerates are less likely to pass through the
cell wall to cause bacterial damage from the interior, and
thus penetration is unlikely the main mechanism for anti-
bacterial activity of CuZnFe oxide NPs. But it can slowly
release metal ions capable of crossing into membranes and
disrupt cellular processes from inside the cell.12,29 Another
potential mechanism that might explain susceptibility of
E. coli to CuZnFe oxide NPs is its higher affinity to LP,36
the main constituent of the outer membrane of gram-negative
bacteria. The physical interaction between CuZnFe oxide
NPs and bacterial cells can disrupt the cell wall structure,
leading to malfunction and to finally bacterial death. Thus, a
bactericidal activity of trimetal oxide NPs can be attributed to
a variety of properties. We assumed that CuZnFe oxide NPs
are characterized differently from individual metal NPs due
to their size and surface chemistry. The exact mechanisms for
antibacterial activity of CuZnFe oxide NPs are still unclear
and more investigation is required.
The individual CuO NPs had a greater impact on E. faecalis
than on E. coli as assessed by cell death analysis (Figure 5).
This is consistent with the findings of Ruparelia et al,7 who
also reported that CuO NPs affect various strains of E. coli
differently. In this matter, they concluded that gram-negative
bacteria may be affected less by the CuO NPs, which might
involve more complex mechanisms. Also, Premanathan et al37
found that gram-negative bacteria can be more resistant to
CuO NPs than gram-positive bacteria. Of the different metals,

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Alzahrani et al
Cu and CuO have been widely used as cheap and effective
bacteriostatic agents for sterilizing liquids and biological tis-
sues.32 It is important to note that some bacterial species are
more sensitive to particular NPs than are others.26,32 Therefore,
CuO NP may be a promising antibacterial agent in combina-
tion with other metallic NPs. By contrast, we used ZnO NPs
and showed a greater effect on the viability of the gram-
negative E. coli than on the gram-positive E. faecalis, a result
that may correspond to the thickness of the cell walls.
The results of CFU measurements indicated that the
action of CuZnFe oxide NPs on E. coli was non-permanent,
by showing recovery of the treated cells (Figure 8). The CFU
results also indicated that the CuZnFe oxide NPs had a similar
effect on E. coli and E. faecalis and an intermediate effect
compared to the effects by the CuO or ZnO NPs. Trimetal
(CuZnFe) oxide NPs affected biofilm formation to a lesser
degree than did individual ZnO and CuO NPs. This indicates
that the reduction in biofilm formation was not a result of
cell loss but of the NP effect. Reduced biofilm formation
with exposure to CuO and ZnO correlated with cell death
and reduced CFU count for both E. faecalis and E. coli, but
this was not the case with CuZnFe oxide NPs. The reduced
disruption of biofilm formation by CuZnFe oxide NPs might
result from the disruption in quorum sensing rather than from
the effect of NPs. Quorum sensing has been established as an
important factor in biofilm formation in both gram-positive
and gram-negative bacteria.38–41 However, more investigation
is needed on the effect of metallic NPs on biofilm inhibi-
tion to fully understand the reason. Also, there is a need to
know how biofilm inhibition occurs and what antibacterial
mechanisms are involved.
To sum up, the implication from the results of our study
is that trimetal (CuZnFe) oxide NPs can be very useful in
terms of antibacterial activity. Overall, the effect of CuZnFe
oxide NPs on E. faecalis and E. coli lies between the effect of
CuO and ZnO NPs. Therefore, we assume that the cytotoxic-
ity of CuZnFe oxide NPs may be also between that of pure
ZnO and CuO nanoparticles. This might be explained by the
buffering caused by the presence of ferric oxide (Fe2O3) in
the trimetal NPs, leading to a reduced toxic effect. Also, it
has to be emphasized that the presence of Fe2O3 NPs causes
less toxicity than other metal oxide NPs, as previously sug-
gested by Azam et al.17 However, further investigations are
needed to determine whether these hypotheses are true or not.
Finally, with the current drug-delivery techniques, CuZnFe
oxide NPs could be administered to the infected site for in
situ treatment. These NPs could also be used as coating agents
for various medical devices to prevent attachment and growth
of a wide range of pathogenic bacteria.
86 submit your manuscript | www.dovepress.com
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Conclusion
The antimicrobial effect of the trimetal (CuZnFe) oxide
NPs on both gram-positive and gram-negative bacteria
was studied and compared with that of ZnO and CuO NPs.
The CuZnFe oxide NPs were characterized using XRD,
HRTEM, and EDX. HRTEM images showed that the NPs
are almost spherical with a tendency to agglomerate. On
the basis of several HRTEM images taken at different
angles, the average particle size of CuZnFe oxide NPs was
estimated to be 42±2 nm. Analysis of E. coli cell death
demonstrated that CuZnFe oxide NPs have greater anti-
bacterial efficiency than pure ZnO and CuO NPs. On the
other hand, the effect of CuZnFe oxide NPs on E. faecalis
was between that of ZnO and CuO. The monitoring of
biofilm formation by both bacterial cells in the presence
of the NPs used in these experiments revealed that the
efficiency of CuZnFe oxide NPs in inhibiting biofilm for-
mation is comparable to that of pure ZnO and CuO NPs.
However, the number of CFUs showed that the effect of
CuZnFe oxide NPs on cell viability was between that of
the ZnO and CuO NPs.
Acknowledgments
This work was supported by King Saud University, Deanship
of Scientific Research, College of Science Research Center,
Kingdom of Saudi Arabia.
Author contributions
KEA, HSA, and AAN designed the study and the experi-
ments, and wrote the manuscript. HSA and AAN carried
out the biological part of the experiments. KEA, RW,
AMA, and AME synthesized and characterized the NPs. All
authors helped discuss, interpret, and shape the research and
the manuscript, contributed toward data analysis, drafting
and critically revising the paper, gave final approval of the
version to be published, and agree to be accountable for all
aspects of the work.
Disclosure
The authors report no conflicts of interest in this work.
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